No abstract

Nonreceptor tyrosine kinase Abl is an actin-binding protein and a key regulator of neuronal axonal development. Although Abl family kinases also are localized in dendrites and are implicated in postsynaptic functions, it is not clear how Abl kinases regulate dendritic morphogenesis. Using a developing hippocampal culture as a model, we found that the inhibition of Abl kinases by STI571 leads to a remarkable simplification of dendritic branching similar to the phenotype caused by an increased activity of small GTPase RhoA. Time-lapse microscopic imaging reveals a prominent reduction of dendritic branching. In contrast, neurons expressing a constitutively active v-abl construct (CA-Abl) show an exuberant microtubule-associated protein 2-positive (MAP2-positive) dendrite outgrowth, suggesting that Abl modulates dendritic growth. Biochemical assays using a glutathione S-transferase pull-down method to determine GTP-bound active Rho GTPases demonstrate that Abl inhibition increases RhoA activity but has no effect on the activity of Rac1 or Cdc42. At the cellular level the alteration of Abl also changes actin organization consistent with RhoA inhibition. Suppression of the RhoA downstream effector Rho kinase reverses STI571-induced dendritic simplification, demonstrating that activity of the Rho pathway is responsible for the Abl-induced changes in dendrogenesis. Furthermore, CA-Abl-induced neurite outgrowth is blocked by the expression of a constitutively active RhoA construct. The CA-Abl phenotype is not affected by destabilization of microtubules but is reversed partially when actin filaments are stabilized with jasplakinolide. Together, these studies support a critical role for Abl kinases in regulating dendrogenesis by inducing actin cytoskeletal rearrangements in cooperation with Rho GTPases.

No abstract

Body axes and germ layers evolve at gastrulation, and in mammals are driven by many genes; however, what orchestrates the genetic pathways during gastrulation remains elusive. Previously, we presented evidence that microRNA-17 (miRNA-17) family members, miR-17-5p, miR-20a, miR-93, and miR-106a were differentially expressed in mouse embryos and functioned to control differentiation of the stem cell population. Here, we identify function(s) that these miRNAs have during gastrulation. Fluorescent in situ hybridization miRNA probes reveal that these miRNAs are localized at the mid/posterior primitive streak (ps) in distinct populations of primitive ectoderm, mesendoderm, and mesoderm. Seven different miRNA prediction algorithms are identified in silico bone morphogenic protein receptor 2 (Bmpr2) as a target of these miRNAs. Bmpr2 is a member of the TGFβ pathway and invokes stage-specific changes during gastrulation. Recently, Bmpr2 was shown regulating cytoskeletal dynamics, cell movement, and invasion. Our previous and current data led to a hypothesis by which members of the miR-17 family influence gastrulation by suppressing Bmpr2 expression at the primitive streak. This suppression influences fate decisions of cells by affecting genes downstream of BMPR2 as well as mesoderm invasion through regulation of actin dynamics.

Presenilin mutations are linked to the early onset familial Alzheimer’s disease (FAD) and lead to a range of neuronal changes, indicating that presenilins interact with multiple cellular pathways to regulate neuronal functions. In this report, we demonstrate the effects of FAD-linked presenilin 1 mutation (PS1M146L) on the expression and distribution of filamin, an actin cross-linking protein that interacts with PS1 both physically and genetically. By using immunohistochemical method, we evaluated hippocampal dentate gyrus for alterations of proteins involved in synaptic plasticity. Among many proteins expressed in the hippocampus, calretinin, glutamic acid decarboxylase (GAD67), parvalbumin, and filamin displayed distinct changes in their expression and/or distribution patterns. Striking anti-filamin immunoreactivity was associated with the polymorphic cells of hilar region only in transgenic mice expressing PS1M146L. In over 20% of the PS1M146L mice, the hippocampus of the left hemisphere displayed more pronounced upregulation of filamin than that of the right hemisphere. Anti-filamin labeled the hilar neurons only after the PS1M146L mice reached after four months of age. Double labeling immunohistochemical analyses showed that anti-filamin labeled neurons partially overlapped with cholecystokinin (CCK), somatostatin, GAD67, parvalbumin, and calretinin immunoreactive neurons. In cultured HEK293 cells, PS1 overexpression resulted in filamin redistribution from near cell peripheries to cytoplasm. Treatment of CHO cells stably expressing PS1 with WPE-III-31C or DAPT, selective γ-secretase inhibitors, did not suppress the effects of PS1 overexpression on filamin. These studies support a γ-secretase-independent role of PS1 in modulation of filamin-mediated actin cytoskeleton.